CLSI Methodology Research Articles

In vitro activity of olorofim against clinical isolates of Scedosporium species and Lomentospora prolificans using EUCAST and CLSI methodologies.

To evaluate the in vitro activity of olorofim, a new broad-spectrum antifungal with a novel mechanism of action, against a collection of 123 Spanish clinical isolates belonging to five Scedosporium species and Lomentospora prolificans. The activity of olorofim against Scedosporium apiospermum (n = 30), Scedosporium boydii (n = 30), Scedosporium ellipsoideum (n = 10), Scedosporium aurantiacum (n = 20), Scedosporium dehoogii (n = 3) and Lomentospora prolificans (n = 30) was compared with that of amphotericin B, voriconazole, isavuconazole and micafungin by performing EUCAST and CLSI reference methods for antifungal susceptibility testing. Amphotericin B and isavuconazole showed MICs ≥2 mg/L for all the species evaluated and voriconazole was moderately active (GM, MIC50 and MIC90 values ≤2 mg/L) against all of them except L. prolificans. Micafungin was effective against S. apiospermum complex strains, but exhibited elevated MECs for S. dehoogii and S. aurantiacum. Olorofim showed low MICs for all the Scedosporium strains tested (GM values were lower than 0.130 and 0.339 by the EUCAST method and the CLSI method, respectively, for all of the species), including those belonging to the MDR species L. prolificans, for which GM values were 0.115 and 0.225 mg/L by the EUCAST method and the CLSI method, respectively, while the GMs for the rest of the antifungals evaluated were higher than 3.732 mg/L using both methodologies. Olorofim displayed promising in vitro activity against the Scedosporium and L. prolificans strains tested, some of which have reduced susceptibility to the antifungals that are currently in use.

In vitro activity of the siderophore cephalosporin, cefiderocol, against molecularly characterized, carbapenem-non-susceptible Gram-negative bacteria from Europe.

ObjectivesMany carbapenem-resistant (CR) Gram-negative (GN) pathogens exhibit MDR, meaning few therapeutic options are available for CR-GN infections. Cefiderocol, a siderophore cephalosporin, has demonstrated in vitro efficacy against CR-GN bacteria. In the SIDERO-CR-2014–2016 surveillance study, European clinical isolates comprising carbapenem-non-susceptible (CarbNS) Enterobacterales and MDR non-fermenters were tested against cefiderocol and comparators.MethodsCefiderocol MICs were determined using iron-depleted CAMHB, and comparators using CAMHB, per recommended CLSI methodology. Carbapenemase gene profiles were determined using PCR.ResultsIsolates (N = 870) from 23 European countries comprised CarbNS Enterobacterales (n = 457), MDR Pseudomonas aeruginosa (n = 177) and MDR Acinetobacter baumannii (n = 236). The most common carbapenemases were KPC (52%), OXA-48-like (19%), VIM (14%) and NDM (8%) in Enterobacterales, VIM (41%) in P. aeruginosa and OXA-23-like (57%) and OXA-24/40-like (37%) in A. baumannii. Most carbapenemase-producing isolates (65%) co-carried ESBLs. Approximately half of P. aeruginosa isolates were negative for carbapenemases, compared with 10% of Enterobacterales and 3% of A. baumannii. A similar proportion of Enterobacterales were susceptible to cefiderocol (81.6%; 79.0% of VIM producers; 51.4% of NDM producers; based on EUCAST breakpoint values) compared with comparator antimicrobial agents, including colistin (76.4%; 93.5% of VIM producers; 78.4% of NDM producers) and ceftazidime/avibactam (76.6%; 1.6% of VIM producers; 2.7% of NDM producers). Of P. aeruginosa isolates, 98.3% were susceptible to cefiderocol (100% of VIM producers), similar to colistin (100%). Against A. baumannii, 94.9% had cefiderocol MIC ≤2 mg/L and 93.6% of isolates were susceptible to colistin.ConclusionsCefiderocol demonstrated potent activity against CarbNS and MDR GN bacteria, including non-fermenters and a wide variety of MBL- and serine-β-lactamase-producing strains.

Oral prevalence and antifungal susceptibility of Candida species in cystic fibrosis patients

ObjectiveThis study aimed at assessing the oral prevalence ofCandida species in cystic fibrosis patients and the antifungal susceptibility of the isolates. DesignOne hundred patients aged 3–20 years old were included in the study and were divided into three groups: G1 (low severity disease): 25 cystic fibrosis patients with Shwachman-Kulczycki score (SK) between 100 and 71; G2 (high severity disease): 25 cystic fibrosis patients with SK score under 40; and G3 (control): 50 healthy patients age- and gender-matched to cystic fibrosis patients. Stimulated saliva samples were collected and the oral fungal concentrations were assessed. Isolates were identified by phenotypic and genotypic tests. Antifungal susceptibilities to amphotericin B, flucytosine and fluconazole were determined by CLSI methodology. Fungal counts were compared by Kruskal Wallis and Dunn’s test (5%). ResultsA total of 68 % of Group 1, 80 % of Group 2, and 44 % of controls yielded positive Candida cultures. Oral concentrations of fungi were significantly higher in cystic fibrosis patients in relation to the control group (p < 0.0005). No significant difference was observed between low and high severity cystic fibrosis groups (p > 0.05). C. albicans was most frequently isolated species in all groups. Higher variability of Candida species was observed in the control group. C. dubliniensis and C. tropicalis were only detected among cystic fibrosis groups. All the isolates were susceptible to flucytosine and fluconazole. ConclusionsPatients with cystic fibrosis were more frequently colonized by Candida species and showed higher oral fungal burden. No antifungal resistant isolates were detected.

MIC distributions for amphotericin B, fluconazole, itraconazole, voriconazole, flucytosine and anidulafungin and 35 uncommon pathogenic yeast species from the UK determined using the CLSI broth microdilution method.

Epidemiological cut-off values and clinical interpretive breakpoints have been developed for a number of antifungal agents with the most common Candida species that account for the majority of infections due to pathogenic yeasts species. However, less-common species, for which susceptibility data are limited, are increasingly reported in high-risk patients and breakthrough infections. The UK National Mycology Reference Laboratory performs routine antifungal susceptibility testing of clinical yeast isolates submitted from across the UK. Between 2002 and 2016, >32 000 isolates representing 94 different yeast species were referred to the laboratory. Here we present antifungal susceptibility profiles generated over this period for amphotericin B, fluconazole, voriconazole, itraconazole, anidulafungin and flucytosine against 35 species of uncommon yeast using CLSI methodologies. MIC data were interpreted against epidemiological cut-off values and clinical breakpoints developed with Candida albicans, in order to identify species with unusually skewed MIC distributions that potentially indicate resistance. Potential resistance to at least one antifungal agent (>10% of isolates with MICs greater than the epidemiological cut-off or clinical breakpoint) was evidenced for 29/35 species examined here. Four species exhibited elevated MICs with all of the triazole antifungal drugs against which they were tested, and 21 species exhibited antifungal resistance to agents from at least two different classes of antifungal agent. This study highlights a number of yeast species with unusual MIC distributions and provides data to aid clinicians in deciding which antifungal regimens may be appropriate when confronted with infections with rarer yeasts.

694. In vitro Antibacterial Activity of Sulbactam–Durlobactam (ETX2514SUL) Against 121 Recent Acinetobacter baumannii Isolated from Patients in India

BackgroundThe incidence of infections caused by multidrug-resistant Acinetobacter baumannii is increasing at an alarming rate in Southeast Asia and other parts of the world. Sulbactam (SUL) has intrinsic antibacterial activity against A. baumannii; however, the prevalence of β-lactamases in this species has limited its therapeutic use. Durlobactam (ETX2514, DUR) is a novel β-lactamase inhibitor with broad-spectrum activity against Ambler class A, C and D β-lactamases. DUR restores SUL in vitro activity against multidrug-resistant A. baumannii. Against >3,600 globally diverse, clinical isolates from 2012–2017, addition of 4 mg/L DUR reduced the SUL MIC90 from >32 to 2 mg/L. SUL-DUR is currently in Phase 3 clinical development for the treatment of infections caused by carbapenem-resistant Acinetobacter spp.The goal of this study was to determine the activity of SUL-DUR and comparator antibiotics (amikacin (AMK), ampicillin-sulbactam (AMP-SUL), cefoperazone-sulbactam (CFP-SUL) and meropenem (MEM)) against A. baumannii isolated from hospitalized patients in India.MethodsA total of 121 clinical A. baumannii isolates from multiple hospital settings and infection sources were collected between 2016–2019 from six geographically diverse hospitals in India. Species identification was performed by MALDI-TOF. Susceptibility of these isolates to SUL-DUR (10µg/10µg) and comparator antibiotics was determined by disk diffusion using CLSI methodology and interpretive criteria, except for CFP-SUL, for which resistance was defined using breakpoints from the CFP-SUL package insert.ResultsAs shown in Table 1, resistance of this collection of isolates to marketed agents was extremely high. In contrast, based on preliminary breakpoint criteria, only 11.5% of isolates were resistant to SUL-DUR.ConclusionThe in vitro antibacterial activity of SUL-DUR was significantly more potent than comparator agents against multidrug-resistant A. baumannii isolates collected from diverse sites in India. These data support the continued development of SUL-DUR for the treatment of antibiotic-resistant infections caused by A. baumannii. Disclosures All authors: No reported disclosures.

721. In Vitro Activity of Cefiderocol Against Gram-Negative Clinical Isolates From New York City

BackgroundMultidrug-resistant Gram-negative bacteria have become a serious problem in hospitals worldwide. Cefiderocol (CFDC) is a novel siderophore cephalosporin with activity against a wide range of carbapenemase- and ESBL-producing bacteria. We tested the activity of CFDC against (1) a recent collection of clinical isolates and (2) a separate collection of carbapenem-resistant isolates gathered from NYC hospitals.MethodsSusceptibility testing was performed on isolates of E. coli, K. pneumoniae, Enterobacter spp., P. aeruginosa, and A. baumannii gathered in 2017 from 7 hospitals in Brooklyn, NY. Consecutive unique patient clinical isolates from all sources were collected for a three month period. Testing was also done on a collection of carbapenem-resistant isolates from a similar surveillance study conducted in 2013–2014. MICs were performed with iron-depleted cation-adjusted Mueller–Hinton broth for CFDC and agar dilution for other antibiotics according to CLSI methodology. The provisional CLSI breakpoint (≤4 μg/mL susceptible) was used for CFDC. Cephalosporin-resistant isolates were tested for common carbapenemases by PCR.ResultsThe susceptibility results for CFDC and meropenem for the isolates gathered in 2017 are listed in the Table. All of the Enterobacteriacae were susceptible to CFDC including KPC-possessing E. coli (n = 4), K. pneumoniae (n = 20), and Enterobacter spp (n = 3). 99.6% of P. aeruginosa and 100% of A. baumannii (including 8 with blaOXA-23, 2 with blaOXA-24, and 1 with blaKPC) were susceptible to CFDC. For the collection of carbapenem-resistant isolates gathered in 2013–14, 100% of K. pneumoniae (n = 111), 100% of P. aeruginosa (n = 130), and 90% of A. baumannii (n = 78) were susceptible to CFDC.ConclusionCFDC has excellent in vitro activity against Gram-negative clinical isolates from NYC, including a large collection of carbapenem-resistant Enterobacteriaceae, P. aeruginosa, and A. baumannii. Disclosures All authors: No reported disclosures.

728. Activity of Eravacycline Against Contemporary Gram-Negative Clinical Isolates From New York City Hospitals

BackgroundAntibiotic-resistant Gram-negative bacteria, including KPC-producing Enterobacteriaceae and carbapenem-resistant A. baumannii, have been problematic hospital pathogens in NYC and other areas. Eravacycline (ERV), a fluorocycline antibiotic released in the USA in 2018, has demonstrated in vitro activity against many of these strains. We tested the activity of ERV against a recent collection of clinical isolates from NYC hospitals.MethodsFor a 3-month period in 2017, all unique patient isolates of E. coli, K. pneumoniae, Enterobacter spp., and A. baumannii were collected from 7 hospitals in Brooklyn, NY. MICs were performed by broth microdilution for ERV and Tigecycline (TGC) and agar dilution for other antibiotics according to CLSI methodology. Cephalosporin-resistant isolates were screened by PCR for common carbapenemases.ResultsThe susceptibility results for tetracycline and ERV are listed in the Table. Overall, 95% of the Enterobacteriaceae were inhibited by ≤ 0.5 μg/mL of ERV, the FDA-suggested breakpoint. Of 1,876 isolates of E. coli, 4 possessed KPC. ERV MICs for these 4 isolates were 0.125–0.25 μg/mL. Of 518 isolates of K. pneumoniae, 20 possessed KPC. The ERV MIC50 and MIC90 for these isolates were 1 and 1 μg/mL, respectively. Of 172 isolates of Enterobacter spp., 3 possessed KPC. ERV MICs for these 3 isolates were 0.5–1 μg/mL. Of 45 isolates of A. baumannii, 11 isolates possessed a carbapenemase (OXA23 in 8, OXA24 in 2, and KPC in 1). The ERV MIC50 and MIC90 for these isolates were 1 and 2 μg/mL, respectively. Overall, ERV MICs were two-fold lower than TGC MICs for A. baumannii.ConclusionERV possesses significant in vitro activity against contemporary clinical isolates of Enterobacteriaceae and A. baumannii from NYC, including many carbapenemase producing strains. Disclosures All authors: No reported disclosures.

Impact of the Major Candida glabrata Triazole Resistance Determinants on the Activity of the Novel Investigational Tetrazoles VT-1598 and VT-1161.

VT-1161 and VT-1598 are promising investigational tetrazole antifungals that have shown in vitro and in vivo activity against Candida and other fungi. Candida glabrata is a problematic opportunistic pathogen that is associated with high mortality in invasive infection, as well as both intrinsic and rapidly acquired antifungal resistance. The MICs of VT-1161 and VT-1598 were determined by CLSI methodology to evaluate their in vitro activities against clinical C. glabrata isolates and strains containing individual deletions of the zinc cluster transcription factor genes PDR1 and UPC2A as well as the efflux transporter genes CDR1, PDH1, and SNQ2 Overall, both tetrazoles demonstrated relative activities comparable to those of the tested triazole antifungals against clinical C. glabrata isolates (MIC range, 0.25 to 2 mg/liter and 0.5 to 2 μg/ml for VT-1161 and VT-1598, respectively). Deletion of the PDR1 gene in fluconazole-resistant matched clinical isolate SM3 abolished the decreased susceptibility phenotype completely for both VT-1161 and VT-1598, similarly to the triazoles. UPC2A deletion also increased susceptibility to both triazoles and tetrazoles but to a lesser extent than PDR1 deletion. Of the three major transporter genes regulated by Pdr1, CDR1 deletion resulted in the largest MIC reductions for all agents tested, while PDH1 and SNQ2 deletion individually impacted MICs very little. Overall, both VT-1161 and VT-1598 have comparable activities to those of the available triazoles, and decreased susceptibility to these tetrazoles in C. glabrata is driven by many of the same known resistance mechanisms.

In vitro activity of olorofim (F901318) against clinical isolates of cryptic species of Aspergillus by EUCAST and CLSI methodologies.

To investigate the in vitro activity of olorofim (F901318), a novel broad-spectrum antifungal agent, against 150 strains belonging to 16 different cryptic species of Aspergillus by EUCAST and CLSI methodologies. Olorofim, amphotericin B, micafungin, posaconazole and voriconazole were tested against cryptic species belonging to Aspergillus fumigatus complex (n = 57), Aspergillus ustus complex (n = 25), Aspergillus niger complex (n = 20), Aspergillus flavus complex (n = 20), Aspergillus circumdati complex (n = 15) and Aspergillus terreus complex (n = 13) using EUCAST and CLSI methodologies for broth microdilution susceptibility testing of antifungal agents. Olorofim was the only drug with activity against all cryptic species of Aspergillus tested, including the multiresistant species Aspergillus lentulus, Aspergillus fumigatiaffinis and Aspergillus calidoustus. Geometric means of MICs for olorofim were lower (0.017, 0.015 and 0.098 mg/L, respectively, for EUCAST; and 0.015, 0.015 and 0.048 mg/L, respectively, for CLSI) than for amphotericin B (4.438, 12.699 and 0.554 mg/L, respectively, for EUCAST; and 0.758, 1.320 and 0.447 mg/L, respectively, for CLSI), voriconazole (2.549, 2.297 and 5.856 mg/L, respectively, for EUCAST; and 2.071, 1.741 and 5.657 mg/L, respectively, for CLSI) and posaconazole (0.307, 0.308 and 12.996 mg/L, respectively, for EUCAST; and 0.391, 0.215 and 9.514 mg/L, respectively, for CLSI). Olorofim shows encouraging in vitro activity against cryptic species of Aspergillus that can be hard to treat with current antifungal therapies. Further studies are warranted in order to assess its efficacy.

In vitro activity of APX001A against rare moulds using EUCAST and CLSI methodologies

APX001A (E1210) is a novel broad-spectrum antifungal agent that inhibits Gwt1p, a protein that plays an important role in fungal cell wall integrity. Previous studies have shown that APX001A has broad activity against most species of Candida, Aspergillus, Scedosporium, Fusarium and Mucorales. To investigate the in vitro activity of APX001A against 200 isolates belonging to 20 different species of Fusarium, Scedosporium, Lomentospora, Alternaria, cryptic species of Aspergillus and Mucorales. APX001A and comparators were tested using EUCAST and CLSI methodologies for broth microdilution susceptibility testing of antifungal agents. APX001A was generally inactive against Mucorales, but active against all cryptic species of Aspergillus and Scedosporium/Lomentospora species. APX001A shows encouraging in vitro activity against some emerging fungi that are hard to treat with currently available antifungals.

1359. Activity of Meropenem/Nacubactam Combination Against Gram-Negative Clinical Isolates: ROSCO Global Surveillance 2017

BackgroundNacubactam (NAC, OP0595, RG6080) is a novel member of the diazabicyclooctane inhibitor family with a dual mode of action, acting as a β-lactamase inhibitor and an antibacterial agent by means of PBP2 inactivation. NAC restores and extends the activity of β-lactam antibiotics, such as meropenem (MEM), when used in combination against a variety of carbapenem-resistant Enterobacteriaceae (CRE). The first year results of the ROSCO surveillance study for MEM/NAC against contemporary clinical isolates are presented here.MethodsIsolates (n = 4,695) collected in 2017 from 50 sites in the United States and European hospitals included 30 different species of Enterobacteriaceae (EB, n = 3,306), Pseudomonas spp. (n = 960) and Acinetobacter spp. (n = 429). The predominant species of EB are shown in figure below. MICs were determined by broth microdilution following CLSI methodology for MEM/NAC at a fixed 1:1 ratio (w:w) and by titrating MEM with a constant concentration of NAC at 4 mg/L. Results were compared with MIC values of MEM and NAC alone and standard of care antibiotics, including ceftazidime/avibactam (CAZ/AVI).ResultsMIC50/90 for MEM, NAC, and MEM/NAC against all EB isolates and by species are shown in the figure below. NAC alone displayed a bimodal MIC distribution for EB, with a prominent separation at ≤4 mg/L. MEM/NAC 1:1 inhibited 99.5, 99.7, and 99.9% of the 3,306 EB isolates tested, at ≤2, ≤4, and ≤8 mg/L, respectively; while MEM inhibited 96.5, 96.8, and 97.3% of the isolates at the same concentrations. Of 117 (3.5% of total EB) MEM nonsusceptible (by EUCAST) and multidrug resistant (MDR, by Magiorakos AP, et al., 2012) EB, 87.2, 92.3, and 96.6% were inhibited by MEM/NAC 1:1 at ≤2, ≤4, and ≤8 mg/L, respectively. Additionally, MEM/NAC1:1 displayed MIC ≤8 mg/L for 33 out of 37 CAZ/AVI-resistant MDR EB isolates. MEM/NAC had a similar activity to MEM alone against Pseudomonas spp. and Acinetobacter spp. ConclusionMEM/NAC combination shows excellent in vitro activity against current clinical EB isolates and the potential to extend MEM activity to MDR, MEM nonsusceptible and CAZ/AVI-resistant isolates, which supports the continued clinical development of MEM/NAC for infections caused by CREs. This project has been funded in part under HHS BARDA Contract HHSO100201600038C.Disclosures R. Okujava, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland: Employee, Salary. F. Garcia-Alcalde, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland: Employee, Salary. A. Haldimann, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland: Employee, Salary. C. Zampaloni, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland: Employee, Salary. I. Morrissey, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland: Research Contractor, Contracting fee to IHMA. S. Magnet, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland: Research Contractor, Contracting fee to IHMA. N. Kothari, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland: Research Contractor, Contracting fee to IHMA. I. Harding, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland: Research Contractor, Contracting fee to Micron. K. Bradley, Roche Innovation Center Basel, F. Hoffmann-La Roche Ltd., Basel, Switzerland: Employee, Salary.

An Improved Extended-Spectrum-β-Lactamase Detection Test Utilizing Aztreonam plus Clavulanate

Clinical laboratories test for extended-spectrum β-lactamases (ESBLs) for epidemiological and infection control purposes and also for the potential of cephalosporins to cause therapeutic failures. Testing can be problematic, because the CLSI does not recommend the testing of all producers of ESBLs and also falsely negative results may occur with isolates that coproduce AmpC. Boronic acid-supplemented tests can enhance ESBL detection in AmpC producers. Because aztreonam inhibits AmpCs, a study was designed to compare ESBL detection by the CLSI disk test (CLSI), a boronic acid-supplemented CLSI disk test (CLSI plus BA), and an aztreonam plus clavulanate disk test (ATM plus CA). The study tested 100 well-characterized Enterobacteriaceae, Acinetobacter baumannii, and Pseudomonas aeruginosa isolates. Seventy produced TEM, SHV, or CTX-M ESBLs, with 15 coproducing an AmpC and 11 coproducing a metallo-β-lactamase. Thirty ESBL-negative isolates were also tested. Tests were inoculated by CLSI methodology and interpreted as positive if an inhibitor caused a zone diameter increase of ≥5 mm. The percentages of ESBL producers detected were as follows: ATM plus CA, 95.7%; CLSI plus BA, 88.6%; and CLSI, 78.6%. When AmpC was coproduced, the sensitivities of the tests were as follows: ATM plus CA, 100%; CLSI plus BA, 93.3%; and CLSI, 60%. ATM plus CA also detected an ESBL in 90.1% of isolates that coproduced a metallo-β-lactamase. Falsely positive tests occurred only with the CLSI and CLSI plus BA tests. Overall, the ATM plus CA test detected ESBLs more accurately than the CLSI and CLSI plus BA tests, especially with isolates coproducing an AmpC or metallo-β-lactamase.

Triclosan resistance reversion by encapsulation in chitosan-coated-nanocapsule containing α-bisabolol as core: development of wound dressing.

The use of nanoparticles may be particularly advantageous in treating bacterial infections due to their multiple simultaneous mechanisms of action. Nanoencapsulation is particularly useful for lipophilic drugs. In this scenario, triclosan is considered a good candidate due to its lipophilicity, broad-spectrum activity, and safety. In the present study, we have developed and characterized an antimicrobial suspension of triclosan and α-bisabolol against pathogenic strains that are resistant (Pseudomonas aeruginosa) and susceptible (Escherichia coli, Staphylococcus aureus, and Candida albicans) to triclosan. We also aimed to determine the minimum inhibitory concentration, using serial microdilution adapted from a CLSI methodology (Clinical and Laboratory Standards Institute). Challenge test was used to confirm the antimicrobial effectiveness of the nanocapsule formulation, as well as after its incorporation into a commercial wound dressing (Veloderm®). The zeta potential of P. aeruginosa before and after contact with cationic nanocapsules and the ratio between the number of nanocapsules per colony forming unit (CFU) were determined to evaluate a possible interaction between nanocapsules and bacteria. The results showed that nanoencapsulation has improved the antimicrobial activity when tested with two different methodologies. The number of nanocapsules per CFU was high even in great dilutions and the zeta potential was reverted after being in contact with the cationic nanocapsules. The nanocapsules were able to improve the activity of triclosan, even when tested within 28 days and when dried in the wound dressing.

Establishment and Use of Epidemiological Cutoff Values for Molds and Yeasts by Use of the Clinical and Laboratory Standards Institute M57 Standard.

Breakpoints are used to predict whether an antifungal agent will be clinically effective against a particular fungal isolate. They are based on a combination of MIC values, pharmacokinetic/pharmacodynamic values, and clinical outcome data. For many fungus-antifungal combinations, these data might never be available. For these combinations, epidemiological cutoff values (ECVs) provide a methodology for categorizing isolates as either wild type (WT) or non-WT. In this review, we define ECVs, explain how they are generated using the CLSI methodology in standard M57, and describe how they can be used in clinical practice.

Role of inoculum and mutant frequency on fosfomycin MIC discrepancies by agar dilution and broth microdilution methods in Enterobacteriaceae

ObjectivesFosfomycin is re-evaluated as a treatment of multidrug-resistant Enterobacteriaceae infections. However, MIC differences have been described among the different susceptibility testing. The aim was to study the role of the different inoculum size used in agar dilution with respect to broth microdilution, according to CLSI, in the fosfomycin MIC discrepancies. MethodsFosfomycin MICs were determined using agar dilution (reference) and broth microdilution in 220 Escherichia coli (n=81) and Klebsiella pneumoniae (n=139) clinical isolates. Fosfomycin mutant frequencies were determined in 21 E. coli (MIC=1mg/L) and 21 K. pneumoniae (MIC=16mg/L). The emergence of resistant subpopulations of five E. coli strains (MIC=1mg/L) was monitored over the time by microdilution assay using 0, 4 and 8 mg/L of fosfomycin, and eight different inocula (5×105−3.91×103 CFU/well, 1 : 2 dilutions). ResultsFor E. coli, 86.4% of categorical agreement (CA), 9.1% very major errors (VME), 3.3% major errors (ME) and 9.9% minor errors (mE) were found. For K. pneumoniae, CA was 51.1%, VME 15.7%, ME 28.4% and mE 25.2%. Essential agreement (±1–log2) was observed in 55.45%. By microdilution, 35.9% of the MICs showed discrepancies of ≥2 dilutions. Initial inoculum used was 5.63 times higher in the microdilution method, in range with CLSI methodology for both techniques. Fosfomycin mutant frequencies were 6.05×10–5 (4×MIC) to 5.59×10–7 (256×MIC) for E. coli, and 1.49×10–4 (4×MIC) to 1.58×10–5 (16×MIC) for K. pneumoniae. Resistant subpopulations arose mainly after 8 h of incubation with inocula >3.13×104 CFU/well. ConclusionsThe higher inoculum used in the microdilution method enriched the initial inoculum with resistant subpopulations and could partially explain the fosfomycin MIC discrepancies with respect to the agar dilution method.

Fungal Cytological Profiling of Candida albicans Exposed to Diverse Antifungal Agents Including the Novel Gwt1 inhibitor APX001A

BackgroundBacterial cytological profiling accelerates drug discovery efforts by determining the mechanism of action (MOA) of newly developed antibacterial agents. Our goal was to adapt this technology to the identification and study of the MOA of antifungal compounds. Here we explore the utility of Fungal Cytological Profiling (FCP) of C. albicans in revealing changes in morphology over time using for 6 antifungal agents with unique MOA using fluorescently labeled compounds that specifically stain a variety of subcellular structures including DNA and membranes. Included in the analysis was the novel broad spectrum Gwt1 inhibitor APX001A, the active moiety of the prodrug APX001 which is currently in clinical trials for invasive fungal infections.MethodsThe MICs of 6 antifungals vs. C. albicans were determined by CLSI methodology. For FCP, antifungals were added to cultures (1 × 105 cells/mL) in RPMI 1640 (buffered with MOPS) at concentrations near MIC: APX001A (0.064 µg/mL); caspofungin (1 µg/mL); fluconazole (2 µg/mL); flucytosine (2 µg/mL); amphotericin B (1 µg/mL) and nikkomycin (3.33 µg/mL) incubated at 35°C with shaking. At 4 hours and 24 hours, treated cultures were stained with various dyes (for 15 minutes), and staining examined under the fluorescence microscope. Dyes included FM 4–64 (membranes), DAPI (DNA), and Sytox Green (cell viability). High-resolution fluorescence microscopy, image analysis and quantitation of cytological parameters (cell length, width, shape, DNA content) were used to create a cytological profile for each growth condition.ResultsUnique cytological signatures strongly correlated with antifungal MOA: FCZ resulted in rounded cells that lacked hyphal forms; APX001A resulted in abundant intracellular membrane labeling at 4 hours, consistent with an endoplasmic reticulum stress response, with cell death (Sytox Green Staining) at 24 hours.ConclusionFCP is a rapid and accurate method to establish MOA and distinguish between antifungals that inhibit specific biosynthetic pathways (e.g., cell wall) and sub-pathways (glucan vs. chitin synthesis). In addition, this technology can be useful in drug discovery program to determine on-target vs. off-target activity of newly synthesized molecules.Disclosures M. Sharp, Linnaeus: Employee, Salary. Q. Soltow, Amplyx Pharmaceuticals Inc.: Employee, Salary. K. J. Shaw, Amplyx Pharmaceuticals Inc.: Employee, Salary. Linnaeus: Consultant, Consulting fee. J. Pogliano, Linnaeus: Employee, Salary.

Changes in the antibiotic susceptibility of anaerobic bacteria from 2007–2009 to 2010–2012 based on the CLSI methodology

Antimicrobial susceptibility testing of anaerobic isolates was conducted at four independent sites from 2010 to 2012 and compared to results from three sites during the period of 2007–2009. This data comparison shows significant changes in antimicrobial resistance in some anaerobic groups. Therefore, we continue to recommend institutions regularly perform susceptibility testing when anaerobes are cultured from pertinent sites. Annual generation of an institutional-specific antibiogram is recommended for tracking of resistance trends over time.

Emergence of co-production of plasmid-mediated AmpC beta-lactamase and ESBL in cefoxitin-resistant uropathogenic Escherichia coli.

Plasmid-mediated AmpC (pAmpC) and ESBL co-production was detected in Escherichia coli a major etiologic agent of urinary tract infection. Isolates resistant to cefoxitin by CLSI methodology were tested for pAmpC beta-lactamase using phenylboronic acid and ESBLs by combined disk diffusion method. pAmpC/ESBL genes were characterized by PCR and sequencing. Transconjugation experiments were done to study the transfer of pAmpC and ESBL production from clinical isolates as donor to E. coli J53 AziR as recipient. Incompatibility groups of transmissible plasmids were classified by PCR-based replicon typing (PBRT). Among 148 urine culture positive isolates, E. coli was reported in 39.86% (59/148), with 93.22% (55/59) of cefoxitin resistance. pAmpC production was detected in 25, with varied distribution of blaCMY-2 and blaDHA-1type genes alone (n = 13 and 7 respectively) or in combination (n = 5). ESBL co-production was observed in 88% (22/25) of pAmpC producing isolates with predominance of blaTEM (n = 20). Twenty-three transconjugants showed transmission of pAmpC-and ESBL-resistant genes with co-carriage of blaCMY-2 and blaTEM (n = 15) in plasmids of IncF type (n = 9) being predominant, followed by IncI1 (n = 4) and IncH1 (n = 2) in combination. All clinical isolates were clonally diverse. Resistance against different beta-lactams in uropathogenic E. coli has been an emerging concern in resource- poor countries such as India. Knowledge on the occurrence of AmpC beta-lactamases and ESBL amongst this pathogen and its transmission dynamics may aid in hospital infection control.

Effects of Treated versus Untreated Polystyrene on Caspofungin In Vitro Activity against Candida Species.

Significant interlaboratory variability is observed in testing the caspofungin susceptibility of Candida species by both the CLSI and EUCAST broth microdilution methodologies. We evaluated the influence of treated versus untreated polystyrene microtiter trays on caspofungin MICs using 209 isolates of four Candida species, including 16 C. albicans and 11 C. glabrata isolates with defined FKS mutations. Caspofungin MICs were also determined using the commercially available YeastOne and Etest assays and 102 isolates. All C. glabrata isolates had caspofungin MICs of ≥0.5 μg/ml, the clinical breakpoint for caspofungin resistance in this species, measured using trays made of treated polystyrene, regardless of the FKS status. In contrast, susceptible isolates could readily be distinguished from resistant/non-wild-type isolates when caspofungin MICs were measured using untreated polystyrene trays and both the YeastOne and Etest assays. Similar results were also observed for C. krusei isolates, as all isolates had caspofungin MICs above the threshold for resistance measured using treated polystyrene trays. In contrast, C. albicans isolates could be correctly identified as susceptible or resistant when caspofungin MICs were measured with treated or untreated trays and with the YeastOne and Etest assays. MICs falsely elevated above the resistance breakpoint were also not observed for C. tropicalis isolates. These results demonstrated that the use of treated polystyrene may be one factor that leads to falsely elevated caspofungin in vitro susceptibility results and that this may also be a greater issue for some Candida species than for others.

Pharmacodynamics of Isavuconazole in a Dynamic In Vitro Model of Invasive Pulmonary Aspergillosis.

Isavuconazonium sulfate is a novel triazole prodrug that has been recently approved for the treatment of invasive aspergillosis by the FDA. The active moiety (isavuconazole) has a broad spectrum of activity against many pathogenic fungi. This study utilized a dynamic in vitro model of the human alveolus to describe the pharmacodynamics of isavuconazole against two wild-type and two previously defined azole-resistant isolates of Aspergillus fumigatus. A human-like concentration-time profile for isavuconazole was generated. MICs were determined using CLSI and EUCAST methodologies. Galactomannan was used as a measure of fungal burden. Target values for the area under the concentration-time curve (AUC)/MIC were calculated using a population pharmacokinetics-pharmacodynamics (PK-PD) mathematical model. Isolates with higher MICs required higher AUCs in order to achieve maximal suppression of galactomannan. The AUC/MIC targets necessary to achieve 90% probability of galactomannan suppression of <1 were 11.40 and 11.20 for EUCAST and CLSI, respectively.